JP2009525158A - Cohesive bone-forming putty and said putty material - Google Patents

Abstract

  An implantable medical material comprising a malleable, cohesive, shape-retaining putty is disclosed, the implantable medical material comprising inorganic particles, insoluble collagen fibers, and soluble collagen. The medical material of the present invention can be used in combination with a biologically active agent, such as a bone morphogenetic protein for treating bone or other tissue defects in a patient.

Description

  The present invention relates generally to putty that is a medical implant material, and in certain embodiments, to a collagen-containing putty that is a medical implant material.

Various materials have been proposed to treat bone defects. In addition to conventional bone grafts, many synthetic bone graft substitutes have been used, including several putty materials.
In order to effectively cause bone through-growth, the graft material has substantial skeleton-forming substances (eg, biocompatible ceramics and other inorganic skeletons). To derive benefits. Such an inorganic substance is generally a hard and brittle substance. Incorporating substantial amounts of inorganic particles in the putty material has proved difficult (particularly for granules and other relatively large particles). Because the presence of large pieces of hard material disturbs the putty mass, and therefore the putty mass easily collapses or gradually erodes and is necessary for handling prior to transplantation and persistence after transplantation This is because the necessary cohesiveness is lost. This causes migration and separation of the skeletal granulate, thus achieving effective bone growth in the desired volume and achieving effective bone growth from end to end of the desired volume. There are several problems.

  In view of the background in this field, there is a need for an improved putty material that not only incorporates relatively large inorganic particles at a high level, but also maintains a desirable combination of spreadability and agglomeration. In certain embodiments, the present invention relates to these needs.

  Accordingly, in one aspect, the invention relates to an implantable osteogenic medical material comprising a malleable, cohesive, shape-retaining putty comprising a combination of inorganic particles and collagen, wherein the collagen is insoluble collagen. Contains fiber and the same or relatively less weight of soluble collagen. Thus, in one embodiment, the present invention comprises 60-75% by weight aqueous liquid medium and comprises bone morphogenic protein incorporated at a level of about 0.6 mg / cc to about 2 mg / cc. Provides a malleable and cohesive shape-retaining putty. The putty of the present invention contains inorganic particles having an average particle size in the range of 0.4 mm to 5 mm in a dispersed state at a level of 0.25 g / cc to 0.35 / cc in the overall putty. The putty of the present invention further comprises insoluble collagen fibers at a level of 0.04 g / cc to 0.1 g / cc and soluble collagen at a level of 0.01 g / cc to 0.08 g / cc, provided that the putty The weight ratio of the insoluble collagen fibers to the soluble collagen is in the range of 4: 1 to 1: 1.

  In another embodiment, the present invention provides an aqueous liquid medium at 60-75% by weight and 0.25 g / cc to 0.35 g of dispersed inorganic particles having an average particle size in the range of 0.4 mm to 5 mm. An implantable medical material comprising a malleable, cohesive, shape-retaining putty contained at / cc is provided. The putty further comprises insoluble collagen fibers at a level of 0.04 g / cc to 0.1 g / cc and soluble collagen at a level of 0.01 g / cc to 0.08 g / cc, provided that the insoluble collagen fibers and The weight ratio with soluble collagen is in the range of 4: 1 to 1: 1. Such putty can be used as an osteoconductive material (eg, in bone void filler applications) and / or modified to incorporate one or more bone morphogenetic proteins to provide an osteogenic putty. It can also be applied.

  In another embodiment, the present invention provides a method of manufacturing an implantable medical putty material. The method of the present invention includes providing a dry porous material comprising a particulate mineral material having an average particle size of about 0.4 mm to about 5 mm embedded in a collapsible collagen matrix. This dry material is composed of 70-90% by weight of a particulate ceramic material and 10-30% by weight of collagen. The collagen matrix contains insoluble collagen fibers and soluble collagen in a weight ratio of 4: 1 to 1: 1. The method of the present invention further comprises the step of adding an amount of aqueous medium to the dried material and collapsing the collagen matrix to obtain a malleable, coherent shape-retaining putty comprising 60-75% by weight water. Process. In a particular aspect of this embodiment, the aqueous medium contains bone morphogenetic protein in an aqueous medium at a level of about 0.6 mg / cc to about 2 mg / cc so that an implantable osteogenic medical material is obtained. It may be contained in a dissolved state.

  In yet another embodiment, the present invention provides an implant comprising a dry porous body comprising a particulate ceramic material having an average particle size of about 0.4 mm to about 5 mm embedded in a collapsible collagen matrix. Provided, wherein the dry porous body comprises 70-90 wt% particulate mineral material and 10-30 wt% collagen. The collagen matrix includes insoluble collagen fibers and soluble collagen, wherein the insoluble collagen fibers and soluble collagen are present in a weight ratio of 4: 1 to 1: 1. This dry porous body can be wetted with a biocompatible aqueous liquid to form a malleable, cohesive, shape-retaining putty material containing a mixture of collagen fibers, aqueous collagen gel and particulate mineral material. is there.

In yet another embodiment, the present invention provides a method of treating a patient comprising implanting the medical material described herein into the patient.
Still other embodiments and features and advantages of the present invention will be apparent to those skilled in the art from the following description.

(Detailed description of the invention)
In order to facilitate an understanding of the principles of the invention, reference will be made to specific embodiments, and specific terminology will be used to describe these embodiments. Nonetheless, it should be understood that this is not intended to limit the scope of the present invention, and further modifications and improvements to the illustrated apparatus and further applications of the principles of the present invention are intended to It is considered that it is normally considered to those skilled in the art.

  As noted above, in certain aspects, the present invention relates to implantable osteogenic medical patties and methods and materials useful for making such osteogenic medical patties. Preferred medical putty materials of the present invention have a combination of advantageous properties including high mineral content, spreadability, cohesiveness, and shape retention. In this regard, as used herein, “maleable” refers to the ability to permanently change the material from a first shape to a second shape by applying pressure. It means that. As used herein, “cohesive” refers to the ability of an individual putty to be stretched even when stretched, including the ability of the putty to stretch fairly long without breaking when stretched. It means that there is a tendency to remain as a connected mass of. In the context of the putty of the present invention containing insoluble collagen fibers and soluble collagen, when stretched, the preferred putty exhibits stretching, upon which the presence of a substantial amount of interdigitated collagen fibers sticking together is evident. As used herein, “shape-retaining” means that the putty material has a high viscosity and, if no pressure is applied, the putty material tends to retain its shape as it is. It means that. This is in contrast to the less viscous paste-form materials that flow easily and thus become puddles when applied to the surface. In a specific feature of the present invention, a medical putty material that not only contains a considerable amount of large inorganic particles but also exhibits excellent properties in terms of spreadability, cohesiveness, and shape retention by combining the ingredients newly. Is obtained.

  The putty according to aspects of the present invention comprises a combination of soluble and insoluble collagen. “Soluble collagen” refers to the dissolution of individual tropocollagen molecules in an acidic aqueous environment. Tropocollagen can be regarded as a monomeric unit of collagen fibers, and its triple helix structure is clearly understood. As used herein, “insoluble collagen” refers to collagen that cannot be dissolved in an alkaline aqueous solution or any inorganic salt solution without chemical modification. For example, skin, thin skin , And other mammals or reptile coats. For example, “naturally insoluble collagen” is the dermis (the grain side and the flesh side) that is the middle layer of animal skin (eg, bovines and pigs). Can be obtained from. “Regenerated collagen” is essentially a collagen fiber segment that is depolymerized into individual triple helical molecules, exposed to solution, and reconstituted into a fibril-like morphology.

  The collagen putty of a preferred embodiment of the present invention contains soluble collagen and insoluble collagen fibers. Soluble collagen and insoluble collagen fibers can first be made separately and then combined. Soluble collagen and insoluble collagen fibers can be obtained from bovine skin, but can also be made from other collagen sources (eg, bovine tendon, porcine tissue, recombinant DNA technology, and Fermentation).

  In a particular embodiment, the present invention provides a putty form composition comprising insoluble collagen fibers at a level of 0.04 to 0.1 g per cc of putty and soluble collagen at 0.01 to 0.08 g per cc of putty. Offer things. In other embodiments, such compositions comprise insoluble collagen fibers at a level of 0.05 to 0.08 g per cc of putty and soluble collagen at 0.02 to 0.05 g per cc of putty. . In general, the putty of the present invention contributes insoluble collagen fibers in an amount (% by weight) equal to or greater than the amount of soluble collagen in order to contribute in an advantageous manner to the desired handling and grafting properties of the putty material. Including. In a preferred embodiment, the collagen matrix comprises insoluble collagen fibers and soluble collagen in a weight ratio of 4: 1 to 1: 1, more preferably in a weight ratio of about 75:25 to about 60:40. A more desirable putty of the present invention comprises insoluble collagen fibers and soluble collagen in a weight ratio of about 75:25 to about 65:35, and in certain embodiments, a weight ratio of about 70:30.

  The medical putty of the present invention further includes an amount of particulate inorganic material. In a particular embodiment of the invention, the particulate mineral material is present in the putty composition of the invention at a level of at least about 0.25 g per cc of putty, generally from about 0.25 g / cc to about 0.35 g / It is incorporated in the range of cc. Such relatively high levels of inorganic material are useful in providing a skeleton for new bone ingrowth.

  The minerals used in the present invention may include natural or synthetic minerals that are effective to provide a skeleton for bone ingrowth. For example, the inorganic matrix is one from the group consisting of bone fragments, Bioglass®, tricalcium phosphate, biphasic calcium phosphate, hydroxyapatite, coralline hydroxyapatite, and biocompatible ceramics. The above materials can be selected. Biphasic calcium phosphate is a particularly desirable synthetic ceramic for use in the present invention. Such biphasic calcium phosphate is about 50:50 to about 95: 5, more preferably about 70:30 to about 95: 5, more preferably about 80:20 to about 90:10, and most preferably May have a tricalcium phosphate: hydroxyapatite weight ratio of about 85:15. The inorganic material is in the form of particles having an average particle size of about 0.4 mm to about 5.0 mm, more typically about 0.4 mm to about 3.0 mm, and desirably about 0.4 mm to 2.0 mm. It may be a substance.

  In another aspect of the present invention, the inorganic material comprises bone fragments (which may be reticular bone, but preferably is dermal bone) that has been ground to provide an average particle size of those described above with respect to the particulate inorganic material. May include. Both human and non-human bone sources are suitable for use in the present invention, and the bone is essentially an autograft to the mammal to receive the transplant and is allograft. It may be a piece or a xenograft. Appropriate pretreatments known in the art can be applied to minimize the risk of disease transmission and / or immunogenic reactions when bone fragments are used as or in mineral materials.

  In one embodiment, xenogeneic bone that has been pretreated to reduce or eliminate immunogenicity is used in or as a porous mineral matrix in an implant composition. For example, bone can be fired or protein can be removed from the bone to reduce the risk of an immunogenic response to the transplant material.

  The putty form composition of the present invention may comprise a fairly high proportion of a liquid carrier (generally an aqueous liquid such as water, saline or buffer). In one embodiment, the spreadable and cohesive shape-retaining putty of the present invention comprises about 60-75% by weight (preferably about 65-75% by weight) of an aqueous liquid medium (eg, water).

  The putty form composition may further comprise a bone morphogenic protein in an amount effective to render the putty osteogenic when implanted in a mammal (eg, a human patient). In one embodiment, the putty composition of the present invention provides bone morphogenic protein at a level of about 0.6 mg to about 2 mg per cc of putty (preferably about 0.8 mg to about 1.8 mg per cc of putty. Included).

  As mentioned above, the compositions of the present invention generally comprise insoluble collagen fibers and soluble collagen, for example in a weight ratio of about 4: 1 to about 1: 1. The putty composition of the present invention contains more insoluble collagen fibers than soluble collagen (e.g., about 75:25 to about 60:40 by weight, more preferably about 75:25 to about 65:35, and certain In an embodiment, about 70:30) is preferred. Suitable collagenous materials for these purposes can be made using methods known in the literature, or can be produced by industrial sources [eg soluble collagen known as Smed S, known as Smed F. Fiber collagen, and complex collagen known as P1076; manufactured by Kency Nash, Exton, Pa.].

  Any suitable osteogenic material can be used in the methods and / or compositions of the present invention, including, for example, the resulting autologous bone and other suitable osteogenic materials. In certain embodiments, the osteogenic material may include a growth factor that is effective in causing bone formation. The growth factor is desirably a type of protein commonly known as bone morphogenetic protein (BMP), and in certain embodiments may be recombinant human (rh) BMP. These BMP proteins are known to have osteogenic action, chondrogenic action, and other growth / differentiation actions, and include rhBMP-2, rhBMP-3, rhBMP-4 (also called rhBMP-2B), rhBMP. -5, rhBMP-6, rhBMP-7 (rhOP-1), rhBMP-8, rhBMP-9, rhBMP-12, rhBMP-2, rhBMP-13, rhBMP-15, rhBMP-16, rhBMP-17, rhBMP- 18, rhGDF-1, rhGDF-3, rhGDF-5, rhGDF-6, rhGDF-7, rhGDF-8, rhGDF-9, rhGDF-10, rhGDF-11, rhGDF-12, and rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, and BMP-7 are described in U.S. Patent Nos. 5,108,922, 5,013,649, and 5,116. , 738, 5,106,748, 5,187,076, and 5,141,905; BMP-8 is disclosed in PCT Publication WO 91/18098; -9 is disclosed in PCT Publication WO 93/00432; BMP-10 is disclosed in US Pat. No. 5,637,480; BMP-11 is disclosed in US Pat. No. 5,639,638. BMP-12 or BMP-13 is disclosed in US Pat. No. 5,658,882; BMP-15 is disclosed in US Pat. No. 5,635,372; BMP-16 is disclosed in US Disclosed in Patent Nos. 5,965,403 and No. 6,331,612. Other compositions that may be useful include Vgr-2 and any one of the growth / differentiation factors (PCT applications WO94 / 15965, WO94 / 15949, WO95 / 01801, WO95 / 01802, WO94 / 21681, WO 94/15966, WO 95/10539, WO 96/01845, WO 96/02559, and other PCT applications). Further useful in the present invention are BIP disclosed in WO94 / 01557; HP00269 disclosed in JP Publication No. 7-250688; and MP52 disclosed in PCT application WO93 / 16099. The disclosures of all these patents and patent applications are incorporated herein by reference. Also useful in the present invention are the heterodimers and denatured proteins or partial deletion products thereof described above. These proteins can be used alone or in a mixture of two or more. Preferred is rhBMP-2.

  BMP can be produced by genetic recombination techniques or can be purified from a protein composition. BMP may be a homodimer or a heterodimer with another BMP (for example, a heterodimer composed of one BMP-2 monomer and one BMP-6 monomer), Alternatively, heterodimers with other members of the TGF-β superfamily such as activin, inhibin, and TGF-β1 (eg, heterodimers composed of one BMP monomer and one related member of the TGF-β superfamily) Dimer). Examples of such heterodimeric proteins are disclosed, for example, in PCT patent application WO 93/09229, which is hereby incorporated by reference. The amount of bone morphogenetic protein useful in the present invention is an amount effective to stimulate an increased osteogenic effect infiltrating progenitor cells, and the extent and nature of the defect being treated and the carrier and protein used. Depends on several factors including type. In certain embodiments, the amount of bone morphogenetic protein to be supplied ranges from about 0.05 mg to about 1.5 mg.

  Other therapeutic growth factors or therapeutic substances (especially those that can be used to stimulate bone formation) can also be used in the putty of the present invention. Such proteins are known and include, for example, platelet-derived growth factor, insulin-like growth factor, cartilage-derived bone morphogenic protein, growth / differentiation factor [eg, growth / differentiation factor 5 (GDF-5)], and transformed growth There are factors (including TGF-α and TGF-β). Similarly, other biologically derived matrix materials [eg, demineralized bone matrix (DBM)] can be incorporated into the putty of the present invention.

  The bone morphogenetic protein or other biologically active agent used in the present invention can be supplied in the form of a liquid formulation (eg, a buffered aqueous formulation). In certain embodiments, such a formulation is mixed with a dry implant material and received on and / or in a dry implant material to make the osteogenic putty material of the present invention, or Combine with dry transplant material.

  As a further improvement of the composition of the present invention, one skilled in the art will readily appreciate that other bone formation enhancing factors can be incorporated into the composition. Such additional factors include host-compatible osteogenic precursor cells, autologous bone marrow, allogeneic bone marrow, transforming growth factor-β, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, microglobulin-β , Antibiotics, antifungals, wetting agents, glycerol, steroids, and non-steroidal anti-inflammatory compounds.

  In another aspect, the present invention provides a dry implant that can be mixed with an appropriate amount of an aqueous medium to produce the putty material of the present invention. A dry graft material is a porous object comprising a particulate mineral material having an average particle size of about 0.4 mm to about 5 mm embedded in a collapsible collagen matrix. The dry porous graft material comprises 70-90% by weight particulate mineral material and 10-30% by weight collagen. The collagen matrix comprises insoluble collagen fibers and soluble collagen in the above weight ratio (ie 4: 1 to 1: 1), preferably about 75:25 to about 60:40, more preferably about 75:25. At about 65:35, and in certain embodiments at about 70:30. Furthermore, as described above, the granular inorganic material generally has an average particle size of 0.4 mm to 3.0 mm, more preferably an average particle size of 0.4 mm to 2.0 mm.

  The dry porous body has a density of about 0.1 g / cc to about 0.3 g / cc, more preferably a density of about 0.15 g / cc to about 0.25 g / cc, and certain In embodiments, it has a density of about 0.18 g / cc to about 0.25 g / cc. Such dry porous implants further exhibit a porosity of at least about 50%, more desirably a porosity of at least about 70% up to about 90%, and in certain embodiments about 80%. It exhibits a porosity in the range of ~ 90%.

  The dry porous implants of the present invention can be provided in any suitable shape, including cylinders, cubes, or other shapes. In certain embodiments, the dry porous implant can define a reservoir for receiving various amounts of wetting liquid (eg, used in making putty from the dry implant material).

  The dry porous body may be any suitable including, for example, casting a liquid medium containing a dry component and then drying the liquid medium by any suitable means (eg, air drying or freeze drying). Can be manufactured using the method.

  Referring to FIG. 1, a dry porous implant 11 of the present invention is shown. The object 11 has a casting material 12 that defines a reservoir 13. The reservoir 13 has a bottom surface 14 and a side wall 15 and can contain a liquid used to wet the object 11 when the putty is formed (the liquid can be easily charged into the reservoir 13, The liquid can be absorbed into the object 11 as time passes). The object 11 has a top surface 16 that surrounds the reservoir 13, a side wall 17, and a bottom surface 18.

  Referring to FIG. 2, another dry porous implant 21 is shown according to one embodiment of the present invention. The implant 21 is cylindrical in shape and has a circular cross section. Thus, the implant 21 can be conveniently formed in a cylindrical syringe barrel having a cross-sectional dimension that is approximately the same or greater. The implant 21 includes a first end 22, a second end 23, and a smooth, round outer surface.

  In use, a dry porous object (eg, 11, 12) and a sufficient amount of liquid material (eg, an aqueous medium) can be mixed to produce the putty form material described herein. The object (11, 12) exhibits disintegratable properties and can therefore be collapsed by physical manipulation (eg, manual crushing or kneading) to form a putty when mixed with a liquid. Thus, there is generally very little chemical covalent cross-linking (if possible) between collagen substances in the dry material. Other methods that provide integrity to the object (11, 21), such as dehydrothermal cross linking, or cross-linking or adhesion imparted by ionic or hydrogen bonding, can also be used. Therefore, it should be understood that although cross-linking may be present in the dry porous body (11, 21), forming the putty described herein is capable of collapsing the body. This is a characteristic that is left as it is.

  In general, mixing a dry porous implant of the present invention with a liquid carrier and subjecting the resulting mass to physical kneading or other mixing reduces the volume of the dry porous object [eg, , The putty volume will be about 30% to about 70% (more typically about 40% to about 60%) of the volume of the initial implant]. This is due to a break in the initial porosity of the dry implant to form a relatively low porosity or non-porous putty implant composition. Liquid carriers generally include, for example, sterile water, saline, blood, bone marrow, bone marrow fractions and other bone marrow solutions (with and without co-solvents), emulsions, or suspensions. It is an aqueous material that provides sufficient wetting properties so that a putty of 5% is obtained.

  In use, the putty transplant composition of the present invention requires bone growth, for example, to treat the location of a disease, defect, or trauma and / or to promote fixation of an artificial joint. Transplanted to the site. Because the composition of the present invention is in a putty form, it can be positioned, shaped, and / or shaped in voids, defects, or other areas where new bone growth is required. In a particularly advantageous embodiment, the shape-retaining properties of the putty material of the present invention provide sufficient three-dimensional integrity to withstand a considerable degree of compression when abutting the adjacent soft tissue of the body at the bone quality implantation site. Is desirable.

  Bone repair sites that can be treated using the medical putty composition of the present invention include, for example, sites resulting from injuries, defects resulting from surgical procedures, infections, malignant tumors, or developmental malformations. is there. The putty composition of the present invention comprises simple and complex fractures and non-adhesion; external fixation and internal fixation; joint reconstruction such as joint fixation; general arthroplasty; hip joint arthroplasty; femoral head replacement Surgery and humeral head replacement; femoral head surface replacement and total joint replacement; spine repair, including spinal fixation and internal fixation; tumor surgery (eg, filling of defects); discectomy; laminectomy Excision of spinal tumors; anterior cervical and thoracic surgery; spinal injury repair; treatment of scoliosis, kyphosis, and kyphosis; fracture intermaxillary fusion; jaw plastic surgery; temporomandibular joint replacement; Alveolar ridge augmentation and restoration; inlay osteoimplants; transplant placement and modification; sinus lifts; and cosmetic enhancement ment); including such (but not limited to) a wide variety of orthopedic procedures, periodontal surgery, neurosurgical procedures, oral surgery, and can be used in maxillofacial surgery. Specific bones that can be repaired or replaced with an isolate or an implant containing the isolate include, for example, ethmoid bone, frontal bone, nasal bone, occipital bone, parietal bone, temporal bone, mandible, maxilla , Cheekbones, cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacrum, ribs, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, finger bones, iliac bone, sciatic bone, pubic bone, femur, tibia , Ribs, patella, ribs, tarsal bones, and metatarsals.

  The osteogenic putty morphology graft composition of the present invention, once placed in place, has a rate of bone formation as compared to primate patients [eg, smaller mammals (eg, rodents and rabbits)]. Even relatively slow humans] can effectively induce and maintain bone ingrowth to the desired part.

  The osteogenic putty composition of the present invention is particularly advantageous when used on bone or bone parts that are vascularized only to a moderate to low extent. These areas have a much lower rate of bone formation, thus increasing difficulty due to the rapid absorption of the carrier. Examples of sites that are moderately or only marginally vascularized include, for example, the transverse processes or other posterior elements of the spine, long bone shafts (especially the intermediate shafts of the tibia), and cranial defects. A particularly preferred use of the paste composition of the present invention is to promote intervertebral joint fixation in spinal fusion [eg, including interbody posterior and / or interbody lateral fixation] in humans or other mammals. For use as a graft. For example, as described in Example 3 below, an osteogenic putty composition containing recombinant human BMP-2 has been successfully used in posterior lateral fixation procedures and is considered to be highly advantageous. Compared to transplanted bone (which has been considered a “standard” in such fixation for many years), extremely beneficial joint fixation (examined by radiography and biomechanical methods) has been achieved.

  Further, according to another aspect of the present invention, the putty composition of the present invention is a force-bearing spinal transplant device having a compressive strength of, for example, at least about 10,000 N [eg, a fixed cage, dowel, or osteogenic composition. In other devices having pockets, chambers, or other cavities for receiving], onto or around the implant device, in spinal fusion procedures such as interbody fusion Can be used. One example of such an application is described in Example 2 to be described later. According to this example, in order to achieve extremely smooth interbody fixation, the osteogenic putty of the present invention is used between load-bearing bodies. Used in combination with spinal spacers (again, not inferior to autograft bones).

  The medical putty composition of the present invention is further used in combination with a variety of cells, including, for example, progenitor cells and / or stem cells derived from embryonic or adult tissue sources and / or harvested by culture. be able to. For example, the putty of the present invention may be derived from cells (autologous cells) derived from blood, bone marrow, or other tissue sources from the patient to be treated, or cells derived from allogeneic or xenogeneic donor sources. Can be incorporated. In particular embodiments of the present invention, putty of the present invention is, for example, US Patent Publication No. 2005/0130301 by McKay et al., Published June 16, 2005, or US patent filed July 8, 2004. Enhanced bone marrow fractions made as described in application Ser. No. 10 / 887,275, the entire disclosures of which are incorporated herein by reference, can be incorporated. Thus, the putty material is a bone marrow fraction enriched in connective tissue growth components [ie, centrifuging a biological sample and separating the sample into several fractions, including a fraction rich in connective tissue growth components. Can be incorporated. A fraction rich in connective tissue growth components can be isolated from a separated sample, for example, using the fraction in or as a wetting medium for the previously described dry porous objects. Can be incorporated into the putty material of the present invention.

  The present invention further provides a medical kit that can be used to make an implant composition. Such kits can be used to mix dry porous objects according to the present invention with aqueous media and / or other articles such as load-bearing implants (eg spinal spacers) to form a putty. And / or with bone-forming substances such as BMP. In one particular form, such a medical kit includes a dried porous object, a lyophilized form of BMP (eg, rhBMP-2), and an aqueous medium for reconstitution of the BMP to obtain an aqueous formulation. The bone formation putty of the present invention can be prepared by mixing the aqueous medium and the dry porous object.

  In the following, the present invention will be described in more detail with reference to specific examples. Needless to say, these examples are for illustrative purposes only and do not limit the invention.

Example 1
A dry porous cylindrical body weighing 18 cc and weighing about 3.8 g of a putty of the present invention containing rhBMP-2 was added to a buffered aqueous solution of rhBMP-2 [1.5 mg / ml solution, Medtronic Sofarm, Memphis, TN. 9 ml of Danfuk commercially available as INFUSE® bone graft]. The dried porous body was made by casting an aqueous suspension containing insoluble collagen fibers, acid soluble collagen, and ceramic granules, followed by lyophilization. The dry porous body exhibited a porosity of about 85% and was composed of the following components.

  After absorption, the implant material and the added rhBMP-2 solution are mixed thoroughly by kneading to provide approximately 10 cc of implantable putty material (containing approximately 70% water by weight, approximately 0.3 g / cc Biphasic calcium phosphate ceramic granules, 0.05 g / cc insoluble collagen fiber, 0.02 g / cc acid soluble collagen, and 1.5 mg / cc rhBMP-2). The resulting osteogenic putty exhibited excellent properties in handling and use. If no force was applied, the putty retained its shape, formed a malleable and cohesive fibrous mass with the granules taken in, and stretched without breaking even when stretched.

(Example 2)
Use of Putty of the Invention in Interbody Spinal Fixation To achieve interbody fixation 6 months after surgery , using a sheep interbody fusion model, a 5 mm x 11 mm x 11 mm poly stuffed with autograft Ether ether ketone spine spacer [VERTE-STACK® CORNERSTONE® PSR PEEK implant, Medtronic Sofam Danek, Memphis, TN] and the inventive putty of Example 1 The packed PEEK spacer (IP + rhBMP-2) was compared. Blinded radiographic, biomechanical, and histological methods were used to evaluate the effectiveness of these procedures to cause interbody fixation in a sheep lumbar fixation model. Fixation assessment was performed using Faxitron high resolution radiography, non-destructive biomechanical testing, and corresponding undecalcified histology by microradiography. All analyzes were performed by a blinded fashion. In addition, non-decalcified histology was used to assess the bone compatibility of the putty of the present invention. In addition to the treatment groups evaluated, normal spines were also evaluated using the same method. When all data acquisition was complete, the key was broken and the radiographic, biomechanical, and histological data were analyzed by treatment group.

Because of the biomechanical similarity between the lumbar spine of the animal model sheep and the human lumbar spine, the ovine lumbar spine model was used. Wilke et al. Characterized the biomechanical parameters of sheep spine [range of motion, neutral zone, and level stiffness] and have been previously reported by White and Panjabi. Data from human specimens (“Wilke et al., Spine : 22 (20): 2365-2374, 1997”; and “White AA and Panjabi MM, editors, Clinical Biomechanics of the Spine, 2nd edition, J.B. Lippincott, Philadelphia, PA, 1990 "). Wilke et al. “The range of movement of the sheep spine for different loading directions is qualitatively similar to the range of movement of human specimens reported in the literature in terms of their head-to-tail movement.” I found out. They further stated, “Based on the biomechanical similarity between the sheep and human vertebrae demonstrated in this study, the sheep vertebrae may serve as an alternative for evaluating spinal grafts. I think we can do it. ”

Upon arrival at the surgical facility, twelve sheep were housed in an appropriate pasture of a large animal research barn. The insects were removed from the sheep and earmarks were attached for identification. Medical examinations were performed and for animals with signs of respiratory disease, their venous blood was subjected to a complete blood count (CBC).

Sheep were anesthetized. The wool was removed from the dorsal lumbar area and the sheep was placed in a prone position on the operating table.
Iliac crest autograft uptake : Autografts were used as controls. The following protocol was followed. The back and dorsal lumbar area and iliac crest area were rubbed alternately with povidone iodine and isopropyl alcohol multiple times to prepare for aseptic surgery. The area was covered with a cloth and a 3 cm incision was made on the left iliac crest. After partial reflection of the gluteal muscle, a small window was made on the dorsal surface of the iliac crest using only osteotomy. Approximately 2 cc of autologous bone was removed using a curette and then packed into one of the grafts used for lumbar fixation (eg, PEEK spacer) (this is the case for a control). After the administration of morphine sulfate, the iliac crest was terminated. The iliac crest site was closed in the normal manner using 2/0 polysorb for the subcutaneous tissue and stainless steel staples for the skin.

Abdominal ("ventral") interbody fixation : The back and dorsal lumbar areas were rubbed alternately with povidone iodine and isopropyl alcohol multiple times to prepare for aseptic surgery. The area was covered with a cloth, and a retroperitoneal approach was performed on both ventral sides toward L3 / L4 and L5 / L6 from the oblique oblique muscles to the flat abdomen to the transverse process.

Insertion of the graft : Place the bone graft from the iliac crest or the bone graft substitute being studied (rhBMP + putty) in a PEEK spacer (approximately 1.5 cc of material) and transplant into the disc space Then, an end plate was prepared.

Wound sutures : Normal sutures were performed on the external fascia (0 polysorb (absorbable suture)), subcutaneous tissue (2/0 polysorb), and skin (2/0 monofilament non-absorbable suture). The surgical time for each animal was usually about 40 minutes. Perioperative antibiotics (cefazoline sodium) were administered. A post-operative radiograph was taken while the sheep were still in general anesthesia.

Aftercare : Immediately after surgery, the sheep are moved from the operating table to a modified wheelbarrow and taken to the X-ray room while still under general anesthesia, where they take X-ray pictures of the dorsal and lateral directions of the fixation site. It was. After evaluation by radiograph and while in the improved wheelbarrow, the sheep were observed until the swallowing reflex returned. At that time, he was extubated from the sheep and carried to the trailer, where he was in a hungry posture with his chest on the underside. At the end of the day, all animals operated on that day were moved to the research pasture. She was housed outdoors (with shelters in three directions) and allowed to move freely to recover. Postoperative analgesia was performed as described above. Three months after the operation, the sheep was anesthetized and radiographed.

Euthanasia : Twelve sheep were euthanized humanely 6 months after surgery. Euthanasia was performed according to the guidelines described by the “AVMA Panel on Euthanasia” (J. Am. Vet. Med. Assoc., 202: 229-249, 1993). X-ray photographs of the lumbar fixation sites were taken on these sheep to evaluate the degree of fixation in L3-L4.

Collection and handling of test specimens : After euthanasia, all 12 animals were subjected to a thorough macroscopic examination. Conventional gross examination of all major organ systems and histopathological evaluation of pathological disorders (if any) were performed. Animals that died or were euthanized during the course of the study were thoroughly examined to determine the cause of the disease or death. A fixed lumbar spine was obtained by autopsy.

Material analysis : All samples from the lumbar area of the sheep were subjected to mechanical testing for fixation sites. Samples were tested for stiffness against sagittal and frontal bending moments (bending, stretching, right lateral bending, and left lateral bending). Since these mechanical tests are non-destructive tests, the fixation site could also be examined histologically.

Graft material : Twelve treated spine levels (L4-L5) were evaluated. The experimental groups are shown below.

After the survival phase experiment was completed, the spine was frozen immediately for evaluation.
Analysis method 1. In vitro biomechanical testing of treated lumbar motion segments Flexibility testing After thawing frozen specimens, unconstrained biomechanical testing was performed in a non-destructive manner on all vertebrae. All tests were performed within 12 hours after thawing of the specimens. The specimen was frozen only once. Prior to biomechanical testing, instruments applied to the front of the vertebral body were removed so that only the spine and fixed mass structure were tested (not instruments). The flexibility of the motion segment was investigated with respect to bending, stretching, right lateral bending, left lateral bending, right axis rotation, and left axis rotation. The purpose of the biomechanical test is to quantify the stiffness of the lumbar motion segment, which is increased by the conventionally described interbody fusion procedures. The treated (L4-L5) motion segment from the incorporated lumbar spine was dissected to remove extraneous soft tissue, thereby leaving the ligament tissue and bone tissue intact. Specially designed load and support frames were secured to the L4 and L5 vertebrae, respectively.

  In each loading direction, moments of 0 Nm, 0.5 Nm, 2.5 Nm, 4.5 Nm, 6.5 Nm, and 8.5 Nm were obtained. Static loads were used to apply pure moments. Three markers that reflect infrared rays were attached to each vertebra. The position of the infrared reflective marker was recorded using three VICON cameras (ViconPeak, Oxford, UK). Three-dimensional load-displacement data was acquired using simple moments applied in bending, stretching, left lateral bending, right lateral bending, left axis rotation, and right axis rotation. The basic principle of a 3-D motion analysis system for examining 3-D load-displacement behavior is well known in the literature.

  Previously obtained biomechanical data from the normal (untreated) intact group of sheep lumbar motion segments was used as the basic data for normal lumbar motion for sheep L4-L5. The differences in stiffness between the injury group and the normal group were statistically compared. Biomechanical data were analyzed using the Kruskal-Wallis test and the Mann-Whitney test regardless of parameters.

2. X-ray evaluation:
Radiographs were taken immediately after surgery (abdominal dorsal and lateral photographs), at regular postoperative intervals (abdominal dorsal and lateral photographs), and at the time of sacrifice (abdominal dorsal and lateral photographs). High-resolution radiography equipment (Faxtron, Hewlett-Packard Company, McMinnville, Oregon) and high-resolution film (EKTASCAN B / RA film 4153, Kodak Company, Rochester, NY) were used after biomechanical testing. High resolution abdominal dorsal and lateral radiographs of the lumbar spine were obtained. Radiographs were scanned using image analysis software (Image Pro Plus Software version 5.0, Media Cybernetics, Silver Spring, Md.) Operating on a Windows XP workstation. X-ray digital images were obtained using a video camera (model DFC280, Leica Microsystems, Cambridge, UK). In addition, these radiographs were used to gloss samples for histological analysis as described below.

Three blinded assessors evaluated the Faxtron radiographs obtained for interbody fixation. For lateral radiographs, not only the center of the disk space but also the leading and trailing edges were evaluated for fixation based on the following scoring method: 4 = continuous bone bridge formation, 3 = increased bone density, 2 = translucent and some bone bridge formation, and 1 = not fixed. Finally, based on the ventral dorsal and lateral radiographs, the blinded assessor graded the overall fixation score for the spine using the following criteria:
3 = Continuous interbody fixation with no X-ray transmission in the interbody space 2 = Highly accurate fixation with interbody space having X-ray transmission 1 = X-ray transmission of disk space is quite high, from top to bottom 2. There is no evidence of bone bridge formation. Non-decalcification histology and microradiography Processing and staining Non-decalcification section All treatment groups were analyzed for spina bifida levels using the non-decalcification method (microradiography and multiple staining). The staining method was used in conjunction with qualitative light microscopy to evaluate the extent of bone bridge formation and fixation associated with autografts or bone graft substitutes packed in PEEK spacers. Using a dyeing method, the degree of fixation adjacent to or within the PEEK spacer, the host response to the PEEK spacer and bone graft substitute material (if any), the interface of the PEEK spacer, the bone graft and Replacement incorporation and bone remodeling in the fixed mass were evaluated.

  After Faxtron radiography, all vertebral levels containing the graft were grossed in the following manner. A band saw was used to create a frontal cut along the entire length of the spinal column at the front of the handle, leaving the anterior tissue intact. The tissue behind the disc space was discarded. The spinal level anterior column was then bisected into mid-sagly to obtain the right and left halves. The overall disk space was left intact. The lower half of the L4 front post adjacent to the treated level was retained. The upper half of the L5 front post adjacent to the treated level was retained. Labeled right sagittal and left sagittal samples from level, fixed in formalin and treated (continuously dehydrated in alcohol, clarified in xylene substitute, and graded catalyzed Embedded in methyl methacrylate).

  After polymerization was completed and the sample was cured, sectioning and dyeing were performed. The block containing the right and left halves of the spinal level treatment surface was dissected in the sagittal plane with a low speed diamond saw (Buehler Isomet, Lake Bluff, Illinois). For all implanted tissue blocks, sagittal sectioning was performed from the center of the spinal level treatment surface to the outer surface of the treatment area. Thus, section # 1 from the “right block” was sampled at the center of the fixed mass, while section # 6 from the “right block” was sampled at the far outer surface of the treatment area. . Weights were used to obtain sections on the order of 300 μm. Approximately 5-10 sections were made in the sagittal plane through each half of the interbody space. If necessary, grinding was performed to obtain the desired thickness. Section thickness was measured using a metric micrometer (Fowler, Japan). The use of fractional staining using triple staining enabled histological differentiation.

  Stained non-decalcified sections were scanned using image analysis software (Image Pro Plus Software version 5.0, Media Cybernetics, Silver Spring, Md.) Running on a Windows XP workstation. A digital image of the stained non-decalcified section was obtained using a video camera (model DFC280, Leica Microsystems, Cambridge, UK).

Section fixation criteria : Non-decalcified sections were evaluated for fixation at the center of the disk space or through the through-throw area, leading edge, and trailing edge of the instrument. These anatomical locations for each section were considered fixed only if continuous bone bridge formation from upper to lower was observed.

Level fixation criteria : Based on all sections evaluated, the following criteria were used to determine whether there was histological fixation in the level. More than 50% of sections [corresponding microradiograms were also analyzed at the same time but did not “count twice” for fixation (not “counted twice”) but showed continuous bone bridge formation Considered the spine level fixed. Partial fixation was considered to be present if less than 50% of the section (corresponding microradiogram) showed continuous bone bridge formation. If none of the sections and the corresponding microradiograms showed continuous bone bridge formation, no fixation was considered present.

For non-decalcified sections from the microradiographically treated lumbar level, a microradiography device (Faxtron, Hewlett-Packard Company, McMinnville, Oregon) and a spectroscopic film (B / RA 4153 film 4153, Kodak Company, Rochester, NY) Used to take X-ray picture. To determine the exposure time, the thickness of these sections was measured using a metric micrometer (Fowler, Japan). The sections were labeled with ultrafine magic ink and the sections were placed on Ektascan B / RA4153 film and exposed to an X-ray source for about 45 seconds at 20 kV and 3 mA for a section thickness of 100 μm, respectively. The film was then developed, fixed, and analyzed for ossification using standard optical microscopy. Micro-radiograms were scanned using image analysis software (Image Pro Plus Software version 5.0, Media Cybernetics, Silver Spring, Md.) Running on a Windows XP workstation. A digital image of the micro radiograph was obtained using a video camera (model DFC280, Leica Microsystems, Cambridge, UK).

Analyzing the sections and microradiograms did the following:
1) Assess the degree of fixation adjacent to the PEEK spacer, bone graft, and replacement implant, the degree of fixation in the PEEK spacer, bone graft, and replacement implant, and the degree of bone remodeling in the fixed mass did;
2) The response of the host to the biomaterial used was investigated; and 3) the PEEK spacer interface was evaluated.

Result 1. Radiographic evaluation In the two treated groups, good radiographic fixation scores were obtained, and the putty + BMP2 + PEEK group of the present invention performed somewhat better than the autograft group (on average 2.4). 2.8, Table 1). In order to investigate whether there is a relationship between the radiographic fixed score and the treatment, a contingency table was created (Table 2) and chi-square analysis was performed. As can be seen from Table 2, the frequency to achieve a fixed score of 3 (continuous fixation) was 56% for the autograft + PEEK group and 83% for the putty + BMP2 + PEEK group of the present invention. It was. The frequency to achieve a fixed score of 2 (fixation with high accuracy) was 83% for the autograft + PEEK group and 100% for the putty + BMP2 + PEEK group of the present invention. Chi-square analysis revealed that the putty + BMP2 + PEEK group of the present invention tended to achieve a higher radiographic fixation score than the autograft + PEEK group.

2. Results from biomechanics The average stiffness in the six loading directions for the two treated interbody fixation groups and the normal intact group is shown in FIGS. Specifically, FIG. 3 is a chart showing the average flexibility in bending load and extension load of a normal intact group and two treatment groups. The difference between the treatment group and the normal intact group was statistically significant in the two loading directions. The difference between the two treatment groups was statistically significant only in flexion. FIG. 4 is a chart showing the average flexibility in the right axis rotation load and the left axis rotation load of the normal undamaged group and the two treatment groups. The difference between the treatment group and the normal undamaged group was statistically significant in the two loading directions, but the difference between the two treatment groups was not statistically significant. FIG. 5 is a chart showing the average flexibility in the right side bending load and the left side bending load of the normal undamaged group and the two treatment groups. The difference between the treatment group and the normal undamaged group was statistically significant in the two loading directions, but the difference between the two treatment groups was not statistically significant.

  On average, the level stiffness of the two treatment groups is 3.1 times (autograft + PEEK group) and 5.4 times (the putty of the present invention + BMP2 + PEEK group) higher than the normal intact group in bending load. Rigidity (p <0.0001) and 4.6 times (autograft + PEEK group) and 6.1 times (putty of the present invention + BMP2 + PEEK group) higher than normal intact group in extension load (P <0.0001). The two treatment groups have a level stiffness 1.7-3.0 times higher than the normal undamaged group in axial rotation (p <0.0004) and 6.3-3 higher than the normal undamaged group in lateral bending load. 9.3 times higher (p <0.0001). Comparing the flexibility of the two treatment groups, the putty + BMP2 + PEEK group of the present invention, on average, was more rigid in all loading directions. A statistically significant difference was seen only at flexural loads (74% higher stiffness, p <0.002).

(Example 3)
Using a sheep posterior fixation model equipped with a device for using the putty of the present invention in posterior fusion, an autograft and [the putty of the present invention + rhBMP-2 of Example 1] (IP + rhBMP-2) However, the ability to perform posterior lateral fixation was evaluated 6 months after surgery. Blinded radiographic, biomechanical, and histological methods were used to evaluate the effectiveness of these treatments in causing posterior lateral fixation in a sheep lumbar fixation model. Fixation evaluation was performed using Faxtron high resolution radiography, non-destructive biomechanical testing, and non-decalcified histology by microradiography. All analyzes were performed by a blinded fashion. In addition, non-decalcified histology was used to assess the bone compatibility of bone graft substitutes. In addition to the treatment groups evaluated, the same method was used to evaluate the biomechanical properties of normal spine. When all data acquisition was complete, the key was broken and the radiographic, biomechanical, and histological data were analyzed by treatment group.

Because of the biomechanical similarity between the lumbar spine of the animal model sheep and the human lumbar spine, the ovine lumbar spine model was used. Wilke et al. Characterize sheep spine biomechanical parameters [range of motion, neutral zone, and level stiffness] and data from human specimens previously reported by White and Panjabi (“Wilke et al., Spine : 22 (20): 2365-2374, 1997 ";and" White AA and Panjabi MM, editors, Clinical Biomechanics of the Spine, 2nd edition, J. B. Lippincott, Philadelphia, Pennsylvania 19 ". )). Wilke et al. “The range of movement of the sheep spine for different loading directions is qualitatively similar to the range of movement of human specimens reported in the literature in terms of their head-to-tail movement.” I found out. They further stated, “Based on the biomechanical similarity between the sheep and human vertebrae demonstrated in this study, the sheep vertebrae may serve as an alternative for evaluating spinal grafts. I think we can do it. ”

Upon arrival at the surgical facility, twelve sheep were housed in an appropriate pasture of a large animal research barn. The insects were removed from the sheep and earmarks were attached for identification. Medical examinations were performed and for animals with signs of respiratory disease, their venous blood was subjected to a complete blood count (CBC).

  Sheep were anesthetized. The wool was removed from the dorsal lumbar area and the sheep was placed in a prone position on the operating table. The dorsal and dorsal lumbar areas were rubbed alternately with povidone iodine and isopropyl alcohol multiple times to prepare for aseptic surgery. The area was covered with a cloth and a back approach to L3-L6 was performed through the back lumbar musculature.

Iliac crest autograft uptake : Autografts were used as controls. The following protocol was followed. The back and dorsal lumbar area and iliac crest area were rubbed alternately with povidone iodine and isopropyl alcohol multiple times to prepare for aseptic surgery. The area was covered with a cloth and a 3 cm incision was made on the left iliac crest. After partial reflection of the gluteal muscle, a small window was made on the dorsal surface of the iliac crest using only osteotomy. Approximately 2 cc of autologous bone was removed using a curette and then packed into one of the grafts used for lumbar fixation (eg, PEEK spacer) (this is the case for a control). After the administration of morphine sulfate, the iliac crest was terminated. The iliac crest site was closed in the normal manner using 2/0 polysorb for the subcutaneous tissue and stainless steel staples for the skin.

Dorsal ("posterior lateral") interbody fusion : The dorsal lumbar area was rubbed alternately with povidone iodine and isopropyl alcohol multiple times to prepare for aseptic surgery. The area was covered with a cloth and a local anesthetic (bupivacaine) was impregnated along the intended incision site to allow a dorsal approach to L3 and L4 and spinous processes.

Approach to transverse process : A 20 cm incision was made in the skin and the paraspinal muscles were separated from the spinous process and lamina. The facet joint and the transverse process between L3 and L4 were exposed.
Instrument and spinal fixation technique : The cortex was peeled and removed bilaterally from the lateral processes of L3 and L4. A bone graft or bone graft substitute from the iliac crest (rhBMP-2 + putty of the present invention) was placed between the transverse processes (approximately 10 cc per side). The sheep were subjected to transpedal screw fixation using screws and rods. At this point in the procedure, a pedicle screw and rod were inserted.

Wound sutures : Normal sutures were performed on the external fascia (0 polysorb (absorbable suture)), subcutaneous tissue (2/0 polysorb), and skin (2/0 monofilament non-absorbable suture). The surgical time for each animal was usually about 50 minutes. Perioperative antibiotics (cefazoline sodium) were administered. A post-operative radiograph was taken while the sheep were still in general anesthesia.

Aftercare : Immediately after surgery, the sheep are moved from the operating table to a modified wheelbarrow and taken to the X-ray room while still under general anesthesia, where they take X-ray pictures of the dorsal and lateral directions of the fixation site. It was. After evaluation by radiograph and while in the improved wheelbarrow, the sheep were observed until the swallowing reflex returned. At that time, he was extubated from the sheep and carried to the trailer, where he was in a hungry posture with his chest on the underside. At the end of the day, all animals operated on that day were moved to the research pasture. She was housed outdoors (with shelters in three directions) and allowed to move freely to recover. Postoperative analgesia was performed as described above. Three months after the operation, the sheep was anesthetized and radiographed.

Euthanasia : Twelve sheep were euthanized humanely 6 months after surgery. Euthanasia was performed according to the guidelines described by the “AVMA Panel on Euthanasia” (J. Am. Vet. Med. Assoc., 202: 229-249, 1993). X-ray photographs of the lumbar fixation sites were taken on these sheep to evaluate the degree of fixation in L3-L4.

Collection and handling of test specimens : After euthanasia, all 12 animals were subjected to a thorough macroscopic examination. Conventional gross examination of all major organ systems and histopathological evaluation of pathological disorders (if any) were performed. Animals that died or were euthanized during the course of the study were thoroughly examined to determine the cause of the disease or death. A fixed lumbar spine was obtained by autopsy.

Material analysis : All samples from the lumbar area of the sheep were subjected to mechanical testing for fixation sites. Samples were tested for stiffness against sagittal and frontal bending moments (bending, stretching, right lateral bending, and left lateral bending). Since these mechanical tests are non-destructive tests, the fixation site could also be examined histologically.

Graft material :
The experimental groups are shown below. Animals from the autograft group died and were excluded from all analyses. Thus, the total number of animals from the autograft group in the results below was reduced to 5.

  After the survival phase animal experiments were completed, the spine was frozen immediately for evaluation. The effectiveness of bone grafts and bone graft substitutes in achieving posterior lateral fixation and bone recovery was performed by radiographic analysis, biomechanical analysis, and histological analysis described below. The experiment was performed by the blinded fashion. After all analysis was completed, the key was broken and radiographic, biomechanical, and histological data were analyzed according to treatment group.

Analysis method :
1. X-ray evaluation:
X-rays were taken immediately after surgery, at regular post-operative intervals, and at the time of sacrifice. Using a Faxtron (Hewlett-Packard Company, McMinnville, Oregon) high-resolution radiography device and high-resolution film (EKTASCAN B / RA film 4153, Kodak Company, Rochester, NY), A high resolution PAX radiograph was obtained. Radiographs were scanned using image analysis software (Image Pro Plus Software version 5.0, Media Cybernetics, Silver Spring, Md.) Operating on a Windows XP workstation. X-ray digital images were obtained using a video camera (model DFC280, Leica Microsystems, Cambridge, UK). In addition, these radiographs were used to gloss samples for histological analysis as described below.

Three blinded evaluators evaluated the faxitron radiographs obtained for the intertransverse process fusion. For PAX radiographs, for the right and left sides of the level, the intercostal space was evaluated for fixation based on the following scoring method: 4 = continuous bone bridge formation, 3 = increased bone density, 2 = translucent and some bone bridge formation, and 1 = not fixed. Based on the right and left PAX radiographs, the blinded assessor graded the overall fixed score for the spinal level using the following criteria:
3 = continuous fixation: continuous lateral protrusion fixing with respect to right and left and no X-ray transparency;
2 = Possible fixation: There can be interlateral protrusion fixation on the right or left, but neither is X-ray transparent. Right or left lateral protrusion space is radiolucent;
1 = No fixation: Isolated bone formation without continuous bone bridge formation from upper to lower on both the right and left sides. X-ray transmission is high, and there is no evidence of lateral protrusion fixing on the right or left.

After breaking the treatment code, the fixed data from the radiograph was statistically analyzed.
2. In vitro biomechanical testing of treated lumbar motion segments
Flexibility Test After thawing frozen specimens, all vertebrae were subjected to unconstrained biomechanical tests in a non-destructive manner. All metal posterior instruments used to stabilize the posterior lateral fixation were removed prior to biomechanical testing so that the stiffness of the spine and fixation mass structure could be tested. The flexibility of the motion segment was investigated with respect to bending, stretching, right lateral bending, left lateral bending, right axis rotation, and left axis rotation. All tests were performed within 12 hours after thawing of the specimens. The specimen was frozen only once. The purpose of the biomechanical test was to quantify the stiffness of the lumbar motion segment, which is increased by previously described fixation procedures. The treated (L4-L5) motor segment from the incorporated lumbar spine was dissected to remove extraneous soft tissue, thereby leaving the ligament tissue and bone tissue intact. Specially designed load and support frames were secured to the L4 and L5 vertebrae, respectively.

  In each loading direction, moments of 0 Nm, 0.5 Nm, 2.5 Nm, 4.5 Nm, 6.5 Nm, and 8.5 Nm were obtained. Static loads were used to apply pure moments. A 6-degree-of-freedom load cell was placed in series with the tested specimen to confirm the applied moment. Three markers that reflect infrared rays were attached to each vertebra. The position of the infrared reflective marker was recorded using three VICON cameras (Vicon Peak, Oxford, UK). Three-dimensional load-displacement data was acquired using simple moments applied in bending, stretching, left lateral bending, right lateral bending, left axis rotation, and right axis rotation. The three-dimensional coordinate data was analyzed to obtain the rotation angle of the upper vertebra with respect to the lower vertebra and the rotation flexibility of each motion segment.

  Previously obtained biomechanical data from the normal (untreated) intact group of sheep lumbar motion segments was used as the basic data for normal lumbar motion for sheep L4-L5. The differences in stiffness between the injury group and the normal group were statistically compared. Biomechanical data were analyzed using the Kruskal-Wallis test and the Mann-Whitney test regardless of parameters.

3. Non-decalcification histology and microradiography
For each of the processing and staining non-decalcification section treatment groups, the non-decalcification method was used to analyze the intercostal process space of the spina bifida (microradiography and multiple staining). The staining method was used in conjunction with qualitative light microscopy to assess the extent of bone bridge formation and fixation associated with autografts and bone graft substitutes. A dyeing method was used to assess host response to bone graft replacement material.

  After Faxtron radiography, all vertebral levels containing the graft were grossed in the following manner. A band saw was used to create a frontal cut along the entire length of the spinal column at the front of the handle, leaving the anterior tissue intact. The front tissue was discarded. The posterior element at the vertebral level was then bisected into mid-sagitally to obtain the right and left halves. Angled cuts in the axial plane were made so that the issues cranial to the cranial transversal processes could be discarded for the right and left sides. Angled cuts in the axial plane were made so that the right side and left side could be discarded by trimming the issues causal to the transversal processes. The left and right intercostal projection space structures are further divided by the sagittal plane, and the left fixed mass central and lateral samples, and the right fixed mass central and lateral samples are divided. Got. The right and left center and side samples were labeled, fixed in formalin, and processed (continuously dehydrated in alcohol, clarified in xylene or xylene substitute, and graded catalyzed Embedded in methyl methacrylate).

  After polymerization was completed and the sample was cured, sectioning and dyeing were performed. Blocks containing transverse processes, grafts or graft substitutes, and tissues of transverse processes space were dissected in the sagittal plane with a low speed diamond saw (Buehler Isomet, Lake Bluff, Illinois). For the embedded central and side tissue blocks described above, sectioning was started from the center of the fixed mass for both the central and side blocks. Thus, section # 1 from the “right side block” was sampled at the center of the fixed mass, while section # 6 from the “right side block” was the far side anatomical surface of the fixed mass. Samples were collected at (tips of lateral protrusions). Similarly, section # 1 from the “right center block” was sampled at the center of the fixed mass, while section # 6 from the “right center block” was the far central anatomical surface of the fixed mass. Samples were taken at (thin layer and facet joint). Weights were used to obtain sections on the order of 300 μm. About 5-10 sections were made in the sagittal plane through each half of the inter-protrusion space. If necessary, grinding was performed to obtain the desired thickness. Section thickness was measured using a metric micrometer (Fowler, Japan). The use of fractional staining using triple staining enabled histological differentiation.

  Stained non-decalcified sections were scanned using image analysis software (Image Pro Plus Software version 5.0, Media Cybernetics, Silver Spring, Md.) Running on a Windows XP workstation. A digital image of the stained non-decalcified section was obtained using a video camera (model DFC280, Leica Microsystems, Cambridge, UK). The non-decalcified histology section and microradiogram for this experiment were scanned with the back located on top of the image. The abdomen side of the section was usually flat and showed two oval transverse processes. Sections were scanned so that the transverse process was located at the bottom (ventral side) of the image. A millimeter scale (a mm. Scale) was scanned at the bottom (ventral side) of the image. I trimmed the image using Microsoft Photo Editor.

Section fixation criteria : Non-decalcified sections were considered fixed if continuous bone bridge formation from upper to lower was observed. Sections were further evaluated as follows when the presence of non-bone tissue eliminated the need for continuous bone bridge formation. For a non-fixed section, the section is A) non-fixed including an incomplete bridge, but new bone is found in more than 50% of the length of the section; or B) an incomplete bridge Including and unfixed, new bone is found in less than 50% of the length of the section;

Right and left level fixation criteria : Based on all sections evaluated, the following criteria were used to determine whether histological fixation was present on the right or left side of the level. A spine level right or left side was considered fixed if more than 50% of the section and corresponding microradiogram showed continuous bone bridge formation. Partial fixation was considered present if less than 50% of sections from the right or left side of the spine level and corresponding microradiograms showed continuous bone bridge formation. If a section from the right or left side of the spine level and the corresponding microradiogram showed no bone bridge formation, no fixation was considered present.

For non-decalcified sections from the microradiographically treated lumbar level, microradiographic equipment (faxtron radiography equipment, Hewlett-Packard Company, McMinnville, Oregon) and spectral films (B / RA 4153 film 4153, Rochester, NY) X-rays were taken using Kodak). To determine the exposure time, the thickness of these sections was measured using a metric micrometer (Fowler, Japan). The sections were labeled with ultrafine magic ink and the sections were placed on Ektascan B / RA4153 film and exposed to an X-ray source for about 45 seconds at 20 kV and 3 mA for a section thickness of 100 μm, respectively. The film was then developed, fixed, and analyzed for ossification using standard optical microscopy. Micro-radiograms were scanned using image analysis software (Image Pro Plus Software version 5.0, Media Cybernetics, Silver Spring, Md.) Running on a Windows XP workstation. A digital image of the micro radiograph was obtained using a video camera (model DFC280, Leica Microsystems, Cambridge, UK).

  Sections and microradiograms were analyzed to: 1) Histological fixation was assessed; 2) Host response to autograft and bone graft substitute was examined; and 3) Transverse The quality and quantity of bone in the fixed mass in the interprotrusive space was evaluated.

Result 1. Radiographic results Table 3 shows the radiographic average fixed scores for the treated groups. The putty + rhBMP-2 group of the present invention achieved an average fixed score of 2.7. The average fixed score for the autograft group was 2.2.

  The effect of treatment on the radiographic fixation score was further analyzed. A contingency table was created (Table 4) and chi-square analysis was performed. As can be seen from Table 4, the frequency to achieve a fixed score of 3 (continuous fixation) was 67% for the putty + BMP-2 group of the present invention and 40% for the autograft group. there were. The frequency for achieving a fixed score of 2 (fixed with high accuracy) was 100% for the putty + BMP-2 group of the present invention and 80% for the autograft group. The difference between the putty + BMP-2 group and the autograft group was not statistically significant (p <0.09).

2. Results from Biomechanics Group stiffness for treatment groups is shown in FIGS. 6-8 with respect to axial rotation load, lateral bending load, bending load, and extension load. Specifically, FIG. 6 is a chart showing the average stiffness of an intact animal group and a treated animal group under bending and stretching loads, and FIG. 7 is an intact animal group and treated animal group. Is a chart showing the average stiffness of the treated animal group under axial rotational loading, and FIG. 8 shows the average stiffness of the intact and treated animal groups under lateral bending load. It is a chart. Error bars in these drawings indicate standard deviations.

  Compared to a normal intact spine, the fixed segment from the treatment group was statistically more rigid in all loading directions. A statistical comparison between the two treatment groups showed a slight statistically significant difference in the direction of right lateral bending loading (Kruskal-Wallis test, p <0.003). Furthermore, according to the Mann-Whitney post-hoc test, the flexibility in right lateral bending of the putty + BMP2 treatment group of the present invention was significantly more rigid than the autograft treatment group (87% higher stiffness, p <0. 002).

  Although the present invention has been described in detail with reference to the drawings and embodiments, these are for illustration purposes only, and the present invention is not limited thereto. It should be understood that only the preferred embodiments have been described herein, and all possible variations and modifications based on the gist of the invention should be protected. . Furthermore, any references cited in this specification are indicative of the level of ordinary skill in the art, and the entire disclosures of these references are incorporated herein by reference.

1 is a perspective view of a dry porous object graft material of the present invention including a reservoir for receiving a wetting liquid. FIG. It is a perspective view of the cylindrical dry porous object transplant material of this invention. FIG. 3 is a chart showing the average flexibility in flex and stretch loads for a normal intact group and two treatment groups (described in Example 2). 6 is a chart showing the average flexibility in the right axis rotation load and the left axis rotation load of the normal undamaged group and the two treatment groups (described in Example 2). FIG. 6 is a chart showing the average flexibility in the right lateral bending load and the left lateral bending load of the normal intact group and the two treatment groups (described in Example 2). It is a chart which shows the average rigidity under a bending load and an extension load of an intact animal group and a treatment animal group (as described in Example 3). It is a chart which shows the average rigidity under an axial rotation load of an intact animal group and a treatment animal group (described in Example 3). It is a chart which shows the average rigidity under a transverse bending load of an intact animal group and a treatment animal group (described in Example 3).

Claims (10)

  An implantable medical material comprising a malleable, coherent, shape-retaining putty, wherein the putty is a 60-75 wt% aqueous liquid medium; at a level of 0.25 g to 0.35 g per cc of putty, Dispersed mineral particles having an average particle size ranging from 0.4 mm to 5 mm; insoluble collagen fibers at a level of 0.04 g to 0.1 g per cc of putty; and at a level of 0.01 g to 0.08 g per cc of putty Said implantable medical material, wherein the weight ratio of insoluble collagen fibers to soluble collagen is in the range of 4: 1 to 1: 1.   The implantable medical material of claim 1, wherein the putty comprises about 65% to about 75% water.   The implantable medical material of claim 1, wherein the weight ratio of insoluble collagen fibers to soluble collagen in the putty ranges from about 75:25 to about 60:40.   The implantable medical material according to claim 1, wherein the insoluble collagen fibers are present in the putty at a level of 0.05 g / cc to 0.08 g / cc.   The implantable medical material of claim 1, wherein the soluble collagen is present in the putty at a level of 0.02 g / cc to 0.05 g / cc.   The implantable medical material according to claim 4, wherein the soluble collagen is present in the putty at a level of 0.02 g / cc to 0.05 g / cc.   The implantable medical material of claim 1, wherein the inorganic particles comprise biphasic calcium phosphate.   The implantable medical material of claim 1, wherein the inorganic particles have an average particle size ranging from about 0.4 mm to about 2.0 mm.   The weight ratio of insoluble collagen fibers to soluble collagen is about 75:25 to about 65:35; the inorganic particles have an average particle size ranging from about 0.4 mm to about 4.0 mm; Includes insoluble collagen fibers at a level of about 0.05 g / cc to about 0.08 g / cc; and the putty has soluble collagen at a level of about 0.02 g / cc to about 0.05 g / cc. An implantable medical material according to claim 1 comprising.   The implantable medical material of claim 1, further comprising an osteogenic substance.

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